2′-F RNAs are used widely in studies of ribozymes, the selection of aptamers, and in RNA interference. See, Pallan, P. S., Greene, E. M., Jicman, P. A., Pandey, R. K., Manoharan, M., Rozners, E. and Egli, M. (2011) Unexpected origins of the enhanced pairing affinity of 2′-fluoro-modified RNA. Nucleic Acids Res., 39, 3482-3495; Morrissey, D. V., Lockridge, J. A., Shaw, L., Blanchard, K., Jensen, K., Breen, W., Hartsough, K., Machemer, L., Radka, S., Jadhav, V., et al. (2005) Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Nat. Biotechnol., 23, 1002-1007; Layzer, J. M., McCaffrey, A. P., Tanner, A. K., Huang, Z., Kay, M. A. and Sullenger, B. A. (2004) In vivo activity of nuclease-resistant siRNAs. RNA, 10, 766-771; Khati, M., Schüman, M., Ibrahim, J., Sattentau, Q., Gordon, S. and James, W. (2003) Neutralization of infectivity of diverse R5 clinical isolates of human immunodeficiency virus type 1 by gp120-binding 2′F-RNA aptamers. J. Virol., 77, 12692-12698; Chiu, Y. L. and Rana, T. M. (2003) siRNA function in RNAi: a chemical modification analysis. RNA, 9, 1034-1048; Sabahi, A., Guidry, J., Inamati, G. B., Manoharan, M. and Wittung-Stafshede, P. (2001) Hybridization of 2′-ribose modified mixed-sequence oligonucleotides: thermodynamic and kinetic studies. Nucleic Acids Res., 29, 2163-2170; Ruckman, J., Green, L. S., Beeson, J., Waugh, S., Gillette, W. L., Henninger, D. D., Claesson-Welsh L. and Janjić, N. (1998) 2′-Fluoropyrimidine RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165). Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain. J. Biol. Chem., 273, 20556-20567; and Pieken, W. A., Olsen, D. B., Benseler, F., Aurup, H. and Eckstein, F. (1991) Kinetic characterization of ribonuclease-resistant 2′-modified hammerhead ribozymes. Science, 253, 314-317.
2′-F RNAs have improved properties such as resistance to cleavage by RNase A. See Cuchillo, C. M., Nogués, M. V. and Raines, R. T. (2011) Bovine pancreatic ribonuclease: Fifty years of the first enzymatic reaction mechanism. Biochemistry, 50, 7835-7841.
2′-F RNA can be synthesized enzymatically. See, Ono, T., Scalf, M. and Smith, L. M. (1997) 2′-Fluoro modified nucleic acids: polymerase-directed synthesis, properties and stability to analysis by matrix-assisted laser desorption/ionization mass spectrometry. Nucleic Acids Res., 25, 4581-4588; Friedman, A. D., Kim, D., Liu, R. (2015) Highly stable aptamers selected from a 20-fully modified fGmH RNA library for targeting biomaterials. Biomaterials, 36, 110-123; and Cozens, C., Pinheiro, V. B., Vaisman, A., Woodgate, R. and Holliger, P. (2012) A short adaptive path from DNA to RNA polymerases. Proc. Natl. Acad. Sci. U.S.A., 109, 8067-8072. A standard commercially available enzyme for the preparation of 2′-F RNA is the bacteriophage T7 RNA polymerase Y639F in which tyrosine 639 is replaced with phenylalanine. See Sousa R. and Padilla R. (1995) A mutant T7 RNA polymerase as a DNA polymerase. EMBO J., 14, 4609-4621. The Y639F alteration in T7 RNA polymerase reduces discrimination between non-canonical and canonical nucleoside triphosphates. However such discrimination is still substantial, especially when multiple 2′-modified NTPs or 2′-modified GTP (the strict initiation nucleotide for T7 RNA polymerase) are included in the reaction. See Sousa R. and Padilla R. (1995) A mutant T7 RNA polymerase as a DNA polymerase. EMBO J., 14, 4609-4621; and Padilla R. and Sousa R. (1999) Efficient synthesis of nucleic acids heavily modified with non-canonical ribose 2′-groups using a mutant T7 RNA polymerase (RNAP). Nucleic Acids Res., 27, 1561-1563.